This grant application is focused on the question of how to incorporate the extensive basic knowledge of sickle hemoglobin polymerization into rational therapeutic approaches. 1. We will complete the description of the effect of mixtures on homogeneous and heterogenous nucleation of HbS, and test the description using laser photolysis methods. This will be critical for prediction of the outcome both of gene therapies and drug therapies, and will lay the groundwork for understanding mixtures of liganded and unliganded HbS. 2. As an approach to therapy, we will extend the double nucleation model to include liquid-liquid phase- transitions and their effect on polymer formation. Because of the dramatic effects of crowding (which we have previously measured), the formation of dense liquid regions lowers the Hb concentration elsewhere. This approach offers as much as a tenfold improvement over the therapeutic potential of inducing Fetal hemoglobin, which is the foundation of present hydroxyureatherapy. The effect has already been seen in our preliminary work. Photolysis tests will also determine if nucleation and phase separation are correctly combined. 3. We will continue measurements of fiber flexibility and domain rigidity begun in the last grant period, (a) We are measuring the rigidity of polymer domains by magnetically driven beads and simultaneous optical imagining of the growing domains (b) We are measuring fiber rigidty using patterned photolysis to create bent optical channels through which fibers can grow. A number of protein association diseases are known, though none are so well characterized as that of HbS. As a result, the types of questions that can be raised about the biophysical mechanism have often been raised first for HbS, and their solutions then become available to study other diseases as well. Hence, successes generated here are expected to have useful spin-offs to other protein association problems.